Insect resistant and herbicide tolerant soybean event 9582.814.19.1

ABSTRACT

Soybean event 9582.814.19.1 comprises genes encoding Cry1F, Cry1Ac (synpro), and PAT, affording insect resistance and herbicide tolerance to soybean crops containing the event, and enabling methods for crop protection and protection of stored products.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.13/559,177, filed Jul. 26, 2012, which claims priority to ProvisionalApplication No. 61/511,664, filed Jul. 26, 2011, and ProvisionalApplication No. 61/521,798, filed Aug. 10, 2011, all of which are hereinincorporated by reference in their entireties.

BACKGROUND OF INVENTION

The genes encoding Cry1F and Cry1Ac synpro (Cry1Ac) are capable ofimparting insect resistance, e.g. resistance to lepidopteran insects, totransgenic plants; and the gene encoding PAT (phosphinothricinacetyltransferase) is capable of imparting tolerance to the herbicidephoshpinothricin (glufosinate) to transgenic plants. PAT has beensuccessfully expressed in soybean for use both as a selectable marker inproducing insect resistant transgenic crops, and to impart commerciallevels of tolerance to the herbicide glufosinate in transgenic crops.

The expression of foreign genes in plants is known to be influenced bytheir location in the plant genome, perhaps due to chromatin structure(e.g., heterochromatin) or the proximity of transcriptional regulatoryelements (e.g., enhancers) close to the integration site (Weising etal., Ann. Rev. Genet 22:421-477, 1988). At the same time the presence ofthe transgene at different locations in the genome will influence theoverall phenotype of the plant in different ways. For this reason, it isoften necessary to screen a large number of events in order to identifyan event characterized by optimal expression of an introduced gene ofinterest. For example, it has been observed in plants and in otherorganisms that there may be a wide variation in levels of expression ofan introduced gene among events. There may also be differences inspatial or temporal patterns of expression, for example, differences inthe relative expression of a transgene in various plant tissues, thatmay not correspond to the patterns expected from transcriptionalregulatory elements present in the introduced gene construct. For thisreason, it is common to produce hundreds to thousands of differentevents and screen those events for a single event that has desiredtransgene expression levels and patterns for commercial purposes. Anevent that has desired levels or patterns of transgene expression isuseful for introgressing the transgene into other genetic backgrounds bysexual outcrossing using conventional breeding methods. Progeny of suchcrosses maintain the transgene expression characteristics of theoriginal transformant. This strategy is used to ensure reliable geneexpression in a number of varieties that are well adapted to localgrowing conditions.

It is desirable to be able to detect the presence of a particular eventin order to determine whether progeny of a sexual cross contain atransgene or group of transgenes of interest. In addition, a method fordetecting a particular event would be helpful for complying withregulations requiring the pre-market approval and labeling of foodsderived from recombinant crop plants, for example, or for use inenvironmental monitoring, monitoring traits in crops in the field, ormonitoring products derived from a crop harvest, as well as for use inensuring compliance of parties subject to regulatory or contractualterms.

It is possible to detect the presence of a transgenic event by anynucleic acid detection method known in the art including, but notlimited to, the polymerase chain reaction (PCR) or DNA hybridizationusing nucleic acid probes. These detection methods generally focus onfrequently used genetic elements, such as promoters, terminators, markergenes, etc., because for many DNA constructs, the coding region isinterchangeable. As a result, such methods may not be useful fordiscriminating between different events, particularly those producedusing the same DNA construct or very similar constructs unless the DNAsequence of the flanking DNA adjacent to the inserted heterologous DNAis known. For example, an event-specific PCR assay is described inUnited States Patent Application 2006/0070139 for maize eventDAS-59122-7. It would be desirable to have a simple and discriminativemethod for the identification of soybean event 9582.814.19.1.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to a new insect resistant and herbicidetolerant transgenic soybean transformation event, designated soybeanevent 9582.814.19.1, comprising cry1F, cry1Ac and pat, as describedherein, inserted into a specific site within the genome of a soybeancell. Representative soybean seed has been deposited with American TypeCulture Collection (ATCC) with the Accession No. identified in paragraph[0021]. The DNA of soybean plants containing this event includes thejunction/flanking sequences described herein that characterize thelocation of the inserted DNA within the soybean genome. SEQ ID NO:1 andSEQ ID NO:2 are diagnostic for soybean event 9582.814.19.1. Moreparticularly, sequences surrounding the junctions at bp 1400/1401, andbp 1536/1537 of SEQ ID NO:1, and bp 152/153 of SEQ ID NO:2 arediagnostic for soybean event 9582.814.19.1. Paragraph [00012] belowdescribes examples of sequences comprising these junctions that arecharacteristic of DNA of soybeans containing soybean event9582.814.19.1.

In one embodiment, the invention provides a soybean plant, or partthereof, that is resistant to Pseudoplusia includens (soybean looper)and that has a genome comprising one or more sequences selected from thegroup consisting of bp 1385-1415 of SEQ ID NO:1; bp 1350-1450 of SEQ IDNO:1; bp 1300-1500 of SEQ ID NO:1; bp 1200-1600 of SEQ ID NO:1; bp137-168 of SEQ ID NO:2; bp 103-203 of SEQ ID NO:2; and bp 3-303 of SEQID NO:2, and complements thereof. In another embodiment, the inventionprovides seed of such plants.

In another embodiment, the invention provides a method of controllinginsects that comprises exposing insects to insect resistant soybeanplants, wherein the soybean plants have a genome that contains one ormore sequence selected from the group consisting of bp 1385-1415 of SEQID NO:1; bp 1350-1450 of SEQ ID NO:1; bp 1300-1500 of SEQ ID NO:1; bp1200-1600 of SEQ ID NO:1; bp 137-168 of SEQ ID NO:2; bp 103-203 of SEQID NO:2; and bp 3-303 of SEQ ID NO:2, and complements thereof; which arecharacteristic of the presence of soybean event 9582.814.19.1, tothereby control the insects. Presence of the cry1F v3 (cry1F) and cry1Acsynpro (cry1Ac) genes in soybean event 9582.814.19.1 imparts resistanceto, for example, Pseudoplusia includens (soybean looper), Anticarsiagemmatalis (velvetbean caterpillar), Epinotia aporema, Omoidesindicatus, Rachiplusia nu, Spodoptera frugiperda, Spodoptera cosmoides,Spodoptera eridania, Heliothis virescens, Heliocoverpa zea, Spilosomavirginica and Elasmopalpus lignosellus.

In another embodiment, the invention provides a method of controllingweeds in a soybean crop that comprises applying glufosinate herbicide tothe soybean crop, said soybean crop comprising soybean plants that havea genome containing one or more sequence selected from the groupconsisting of bp 1385-1415 of SEQ ID NO:1; bp 1350-1450 of SEQ ID NO:1;bp 1300-1500 of SEQ ID NO:1; bp 1200-1600 of SEQ ID NO:1; bp 137-168 ofSEQ ID NO:2; bp 103-203 of SEQ ID NO:2; and bp 3-303 of SEQ ID NO:2, andcomplements thereof, which are diagnostic for the presence of soybeanevent 9582.814.19.1. Presence of the pat v6 (pat) gene in soybean event9582.814.19.1 imparts tolerance to glufosinate herbicide.

In another embodiment, the invention provides a method of detectingsoybean event 9582.814.19.1 in a sample comprising soybean DNA, saidmethod comprising:

(a) contacting said sample witha first primer at least 10 bp in length that selectively binds to aflanking sequence within bp 1-1400 of SEQ ID NO:1 or the complementthereof, and a second primer at least 10 bp in length that selectivelybinds to an insert sequence within bp 1401-1836 of SEQ ID NO:1 or thecomplement thereof; and assaying for an amplicon generated between saidprimers; or(b) contacting said sample with a first primer at least 10 bp in lengththat selectively binds to an insert sequence within bp 1-152 of SEQ IDNO:2 or the complement thereof, and a second primer at least 10 bp inlength that selectively binds to flanking sequence within bp 153-1550 ofSEQ ID NO:2 or the complement thereof; and(c) assaying for an amplicon generated between said primers.

In another embodiment, the invention provides a method of detectingsoybean event 9582.814.19.1 comprising:

(a) contacting said sample with a first primer that selectively binds toa flanking sequence selected from the group consisting of bp 1-1400 ofSEQ ID NO:1 and bp 153-1550 of SEQ ID NO:2, and complements thereof; anda second primer that selectively binds to SEQ ID NO:3, or the complementthereof;(b) subjecting said sample to polymerase chain reaction; and(c) assaying for an amplicon generated between said primers.

In another embodiment the invention provides a method of breeding asoybean plant comprising: crossing a first plant with a second soybeanplant to produce a third soybean plant, said first plant comprising DNAcomprising one or more sequence selected from the group consisting of bp1385-1415 of SEQ ID NO:1; bp 1350-1450 of SEQ ID NO:1; bp 1300-1500 ofSEQ ID NO:1; bp 1200-1600 of SEQ ID NO:1; bp 137-168 of SEQ ID NO:2; bp103-203 of SEQ ID NO:2; and bp 3-303 of SEQ ID NO:2, and complementsthereof; and assaying said third soybean plant for presence of DNAcomprising one or more sequences selected from the group consisting ofbp 1385-1415 of SEQ ID NO:1; bp 1350-1450 of SEQ ID NO:1; bp 1300-1500of SEQ ID NO:1; bp 1200-1600 of SEQ ID NO:1; bp 137-168 of SEQ ID NO:2;bp 103-203 of SEQ ID NO:2; and bp 3-303 of SEQ ID NO:2, and complementsthereof.

In another embodiment the invention provides an isolated DNA moleculethat is diagnostic for soybean event 9582.814.19.1. Such moleculesinclude, in addition to SEQ ID NOS: 1 and 2, molecules at least 25 bp inlength comprising bp 1400-1401 of SEQ ID NO:1 and at least 10 bp of SEQID NO:1 in each direction from the bp 1400/1401 junction; amplicons atleast 25 bp in length comprising 152-153 of SEQ ID NO:2 and at least 10bp of SEQ ID NO:2 in each direction from the bp 152/153 junction.Examples are bp 1385-1415 of SEQ ID NO:1; bp 1350-1450 of SEQ ID NO:1;bp 1300-1500 of SEQ ID NO:1; bp 1200-1600 of SEQ ID NO:1; bp 137-168 ofSEQ ID NO:2; bp 103-203 of SEQ ID NO:2; and bp 3-303 of SEQ ID NO:2, andcomplements thereof.

In another embodiment the invention provides a method of controllingpests in soybean grain, seed, or seed meal which comprises includingsoybean event 9582.814.19.1 in said grain, seed, or seed meal asdemonstrated by said grain, seed, or seed meal comprising DNA comprisingone or more sequence selected from the group consisting of bp 1385-1415of SEQ ID NO:1; bp 1350-1450 of SEQ ID NO:1; bp 1300-1500 of SEQ IDNO:1; bp 1200-1600 of SEQ ID NO:1; bp 137-168 of SEQ ID NO:2; bp 103-203of SEQ ID NO:2; and bp 3-303 of SEQ ID NO:2, and complements thereof.

The invention also includes soybean plant cells and plant partsincluding, but are not limited to pollen, ovule, flowers, shoots, roots,and leaves, and nuclei of vegetative cells, pollen cells, seed and seedmeal, and egg cells, that contain soybean event 9582.814.19.1.

In some embodiments, soybean event 9582.814.19.1 can be combined withother traits, including, for example, other herbicide tolerance gene(s)and/or insect-inhibitory proteins and transcription regulatory sequences(i.e. RNA interference, dsRNA, transcription factors, etc). Theadditional traits may be stacked into the plant genome via plantbreeding, re-transformation of the transgenic plant containing soybeanevent 9582.814.19.1, or addition of new traits through targetedintegration via homologous recombination.

Other embodiments include the excision of polynucleotide sequences whichcomprise soybean event 9582.814.19.1, including for example, the palgene expression cassette. Upon excision of a polynucleotide sequence,the modified event may be re-targeted at a specific chromosomal sitewherein additional polynucleotide sequences are stacked with soybeanevent 9582.814.19.1.

In one embodiment, the present invention encompasses a soybeanchromosomal target site located on chromosome 02 between the flankingsequences set forth in SEQ ID NOS:1 and 2.

In one embodiment, the present invention encompasses a method of makinga transgenic soybean plant comprising inserting a heterologous nucleicacid at a position on chromosome 02 between the genomic sequences setforth in SEQ ID NOS:1 and 2, i.e. between bp 1-1400 of SEQ ID NO:1 andbp 153-1550 of SEQ ID NO:2.

Additionally, the subject invention provides assays for detecting thepresence of the subject event in a sample (of soybeans, for example).The assays can be based on the DNA sequence of the recombinantconstruct, inserted into the soybean genome, and on the genomicsequences flanking the insertion site. Kits and conditions useful inconducting the assays are also provided.

The subject invention relates in part to the cloning and analysis of theDNA sequences of the border regions resulting from insertion of T-DNAfrom pDAB9582 in transgenic soybean lines. These sequences are unique.Based on the insert and junction sequences, event-specific primers canbe and were generated. PCR analysis demonstrated that these events canbe identified by analysis of the PCR amplicons generated with theseevent-specific primer sets. Thus, these and other related procedures canbe used to uniquely identify soybean lines comprising the event of thesubject invention.

Seed Deposit

As part of this disclosure at least 2500 seeds of a soybean linecomprising soybean event 9582.814.19.1 were deposited with the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va., 20110. The deposit, ATCC Patent Deposit Designation, PTA-12006, wasreceived by the ATCC on Jul. 21, 2011. This deposit was made and will bemaintained in accordance with and under the terms of the Budapest Treatywith respect to seed deposits for the purposes of patent procedure. Thisdeposit was made and will be maintained in accordance with and under theterms of the Budapest Treaty with respect to seed deposits for thepurposes of patent procedure.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the 5′ DNA flanking border sequence for soybean event9582.814.19.1. Nucleotides 1-1400 are genomic sequence. Nucleotides1401-1535 are a rearranged sequence from pDAB9582. Nucleotides 1536-1836are insert sequence.

SEQ ID NO:2 is the 3′ DNA flanking border sequence for soybean event9582.814.19.1. Nucleotides 1-152 are insert sequence. Nucleotides153-1550 are genomic sequence.

SEQ ID NO:3 is the DNA sequence of pDAB9582, which is annotated below inTable 1.

SEQ ID NO:4 is oligonucleotide primer 81419_FW3 for confirmation of 5′border genomic DNA.

SEQ ID NO:5 is oligonucleotide primer 81419_RV1 for confirmation of 3′border genomic DNA.

SEQ ID NO:6 is oligonucleotide primer 81419_RV2 for confirmation of 3′border genomic DNA.

SEQ ID NO:7 is oligonucleotide primer 81419_RV3 for confirmation of 3′border genomic DNA.

SEQ ID NO:8 is oligonucleotide primer 5′IREnd-01 for confirmation of 5′border genomic DNA.

SEQ ID NO:9 is oligonucleotide primer 5′IREnd-02 for confirmation of 5′border genomic DNA.

SEQ ID NO:10 is oligonucleotide primer AtUbi10RV1 for confirmation of 5′border genomic DNA.

SEQ ID NO:11 is oligonucleotide primer AtUbi10RV2 for confirmation of 5′border genomic DNA.

SEQ ID NO:12 is oligonucleotide primer 3′PATEnd05 for confirmation of 3′border genomic DNA.

SEQ ID NO:13 is oligonucleotide primer 3′PATEnd06 for confirmation of 3′border genomic DNA.

SEQ ID NO:14 is the confirmed sequence of soybean event 9582.814.19.1.Including the 5′ genomic flanking sequence, pDAB9582 T-strand insert,and 3′ genomic flanking sequence.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a plasmid Map of pDAB9582 containing the cry1F, cry1Ac and patexpression cassettes.

FIG. 2 depicts the primer locations for confirming the 5′ and 3′ bordersequence of the soybean event pDAB9582.814.19.1.

FIG. 3 depicts the genomic sequence arrangement in soybean eventpDAB9582.814.19.1

DETAILED DESCRIPTION OF THE INVENTION

Both ends of the soybean event 9582.814.19.1 insertion have beensequenced and characterized. Event specific assays were developed. Ithas also been mapped onto the soybean genome (soybean chromosome 02).The event can be introgressed into further elite lines.

As alluded to above in the Background section, the introduction andintegration of a transgene into a plant genome involves some randomevents (hence the name “event” for a given insertion that is expressed).That is, with many transformation techniques such as Agrobacteriumtransformation, the biolistic transformation (i.e. gene gun), andsilicon carbide mediated transformation (i.e. WHISKERS), it isunpredictable where in the genome a transgene will become inserted.Thus, identifying the flanking plant genomic DNA on both sides of theinsert can be important for identifying a plant that has a giveninsertion event. For example, PCR primers can be designed that generatea PCR amplicon across the junction region of the insert and the hostgenome. This PCR amplicon can be used to identify a unique or distincttype of insertion event.

Definitions and examples are provided herein to help describe thepresent invention and to guide those of ordinary skill in the art topractice the invention. Unless otherwise noted, terms are to beunderstood according to conventional usage by those of ordinary skill inthe relevant art. The nomenclature for DNA bases as set forth at 37 CFR§1.822 is used.

As used herein, the term “progeny” denotes the offspring of anygeneration of a parent plant which comprises soybean event9582.814.19.1.

A transgenic “event” is produced by transformation of plant cells withheterologous DNA, i.e., a nucleic acid construct that includes thetransgenes of interest, regeneration of a population of plants resultingfrom the insertion of the transgene into the genome of the plant, andselection of a particular plant characterized by insertion into aparticular genome location. The term “event” refers to the originaltransformant and progeny of the transformant that include theheterologous DNA. The term “event” also refers to progeny produced by asexual outcross between the transformant and another variety thatincludes the genomic/transgene DNA. Even after repeated back-crossing toa recurrent parent, the inserted transgene DNA and flanking genomic DNA(genomic/transgene DNA) from the transformed parent is present in theprogeny of the cross at the same chromosomal location. The term “event”also refers to DNA from the original transformant and progeny thereofcomprising the inserted DNA and flanking genomic sequence immediatelyadjacent to the inserted DNA that would be expected to be transferred toa progeny that receives inserted DNA including the transgene of interestas the result of a sexual cross of one parental line that includes theinserted DNA (e.g., the original transformant and progeny resulting fromselfing) and a parental line that does not contain the inserted DNA.

A “junction sequence” or “border sequence” spans the point at which DNAinserted into the genome is linked to DNA from the soybean native genomeflanking the insertion point, the identification or detection of one orthe other junction sequences in a plant's genetic material beingsufficient to be diagnostic for the event. Included are the DNAsequences that span the insertions in herein-described soybean eventsand similar lengths of flanking DNA. Specific examples of suchdiagnostic sequences are provided herein; however, other sequences thatoverlap the junctions of the insertions, or the junctions of theinsertions and the genomic sequence, are also diagnostic and could beused according to the subject invention.

The subject invention relates in part to event identification using suchflanking, junction, and insert sequences. Related PCR primers andamplicons are included in the invention. According to the subjectinvention, PCR analysis methods using amplicons that span acrossinserted DNA and its borders can be used to detect or identifycommercialized transgenic soybean varieties or lines derived from thesubject proprietary transgenic soybean lines.

The flanking/junction sequences are diagnostic for soybean event9582.814.19.1. Based on these sequences, event-specific primers weregenerated. PCR analysis demonstrated that these soybean lines can beidentified in different soybean genotypes by analysis of the PCRamplicons generated with these event-specific primer sets. Thus, theseand other related procedures can be used to uniquely identify thesesoybean lines. The sequences identified herein are unique.

Detection techniques of the subject invention are especially useful inconjunction with plant breeding, to determine which progeny plantscomprise a given event, after a parent plant comprising an event ofinterest is crossed with another plant line in an effort to impart oneor more additional traits of interest in the progeny. These PCR analysismethods benefit soybean breeding programs as well as quality control,especially for commercialized transgenic soybean seeds. PCR detectionkits for these transgenic soybean lines can also now be made and used.This can also benefit product registration and product stewardship.

Furthermore, flanking soybean/genomic sequences can be used tospecifically identify the genomic location of each insert. Thisinformation can be used to make molecular marker systems specific toeach event. These can be used for accelerated breeding strategies and toestablish linkage data.

Still further, the flanking sequence information can be used to studyand characterize transgene integration processes, genomic integrationsite characteristics, event sorting, stability of transgenes and theirflanking sequences, and gene expression (especially related to genesilencing, transgene methylation patterns, position effects, andpotential expression-related elements such as MARS [matrix attachmentregions], and the like).

In light of all the subject disclosure, it should be clear that thesubject invention includes seeds available under the ATCC Deposit No.identified in paragraph [0021]. The subject invention also includes aherbicide-tolerant soybean plant grown from a seed deposited with theATCC Deposit No. identified in paragraph [0021]. The subject inventionfurther includes parts of said plant, such as leaves, tissue samples,seeds produced by said plant, pollen, and the like (wherein theycomprise cry1F, cry1Ac, pat, and SEQ ID NOS: 1 and 2).

Still further, the subject invention includes descendant and/or progenyplants of plants grown from the deposited seed, preferably aherbicide-resistant soybean plant wherein said plant has a genomecomprising a detectable wild-type junction sequence as described herein.As used herein, the term “soybean” means Glycine max and includes allvarieties thereof that can be bred with a soybean plant.

This invention further includes processes of making crosses using aplant of the subject invention as at least one parent. For example, thesubject invention includes an F₁ hybrid plant having as one or bothparents any of the plants exemplified herein. Also within the subjectinvention is seed produced by such F₁ hybrids of the subject invention.This invention includes a method for producing an F₁ hybrid seed bycrossing an exemplified plant with a different (e.g. in-bred parent)plant and harvesting the resultant hybrid seed. The subject inventionincludes an exemplified plant that is either a female parent or a maleparent. Characteristics of the resulting plants may be improved bycareful consideration of the parent plants.

An insect resistant/glufosinate-tolerant soybean plant of the subjectinvention can be bred by first sexually crossing a first parentalsoybean plant consisting of a soybean plant grown from seed of any oneof the lines referred to herein, and a second parental soybean plant,thereby producing a plurality of first progeny plants; then selecting afirst progeny plant that is resistant to glufosinate; selfing the firstprogeny plant, thereby producing a plurality of second progeny plants;and then selecting from the second progeny plants a plant that isresistant to glufosinate. These steps can further include theback-crossing of the first progeny plant or the second progeny plant tothe second parental soybean plant or a third parental soybean plant. Asoybean crop comprising soybean seeds of the subject invention, orprogeny thereof, can then be planted.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating, added, exogenous genes. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes.Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated, as is vegetative propagation. Otherbreeding methods commonly used for different traits and crops are knownin the art. Backcross breeding has been used to transfer genes for asimply inherited, highly heritable trait into a desirable homozygouscultivar or inbred line, which is the recurrent parent. The source ofthe trait to be transferred is called the donor parent. The resultingplant is expected to have the attributes of the recurrent parent (e.g.,cultivar) and the desirable trait transferred from the donor parent.After the initial cross, individuals possessing the phenotype of thedonor parent are selected and repeatedly crossed (backcrossed) to therecurrent parent. The resulting parent is expected to have theattributes of the recurrent parent (e.g., cultivar) and the desirabletrait transferred from the donor parent.

Likewise an insect resistant/glufosinate-tolerant soybean plant of thesubject invention can be transformed with additional transgenes usingmethods known in the art. Transformation techniques such asAgrobacterium transformation, the biolistic transformation (i.e. genegun), and silicon carbide mediated transformation (i.e. WHISKERS), canbe used to introduced additional trangene(s) into the genome of soybeanevent 9582.814.19.1. Selection and characterization of transgenic plantscontaining the newly inserted transgenes can be completed to identifyplants which contain a stable integrant of the novel transgene inaddition to cry1F, cry1Ac, pat genes of the subject invention.

The DNA molecules of the present invention can be used as molecularmarkers in a marker assisted breeding (MAB) method. DNA molecules of thepresent invention can be used in methods (such as, AFLP markers, RFLPmarkers, RAPD markers, SNPs, and SSRs) that identify genetically linkedagronomically useful traits, as is known in the art. The insectresistance and herbicide-tolerance traits can be tracked in the progenyof a cross with a soybean plant of the subject invention (or progenythereof and any other soybean cultivar or variety) using the MABmethods. The DNA molecules are markers for this trait, and MAB methodsthat are well known in the art can be used to track theherbicide-resistance trait(s) in soybean plants where at least onesoybean line of the subject invention, or progeny thereof, was a parentor ancestor. The methods of the present invention can be used toidentify any soybean variety having the subject event.

Methods of the subject invention include a method of producing an insectresistant/herbicide-tolerant soybean plant wherein said method comprisesbreeding with a plant of the subject invention. More specifically, saidmethods can comprise crossing two plants of the subject invention, orone plant of the subject invention and any other plant. Preferredmethods further comprise selecting progeny of said cross by analyzingsaid progeny for an event detectable according to the subject inventionand favorable varietal performance (e.g. yield). For example, thesubject invention can be used to track the subject event throughbreeding cycles with plants comprising other desirable traits, such asagronomic traits, disease tolerance or resistance, nematode tolerance orresistance and maturity date. Plants comprising the subject event andthe desired trait can be detected, identified, selected, and quicklyused in further rounds of breeding, for example. The subject event/traitcan also be combined through breeding, and tracked according to thesubject invention, with further insect resistant trait(s) and/or withfurther herbicide tolerance traits. Embodiments of the latter are plantscomprising the subject event combined with the aad-12 gene, whichconfers tolerance to 2,4-dichlorophenoxyacetic acid andpyridyloxyacetate herbicides, or with a gene encoding resistance to theherbicide dicamba.

Thus, the subject invention can be combined with, for example, traitsencoding glyphosate resistance (e.g., resistant plant or bacterialEPSPS, GOX, GAT), glufosinate resistance (e.g., pat, bar), acetolactatesynthase (ALS)-inhibiting herbicide resistance (e.g., imidazolinones[such as imazethapyr], sulfonylureas, triazolopyrimidine sulfonanilide,pyrmidinylthiobenzoates, and other chemistries [Csr1, SurA, et al.]),bromoxynil resistance (e.g., Bxn), resistance to inhibitors of HPPD(4-hydroxlphenyl-pyruvate-dioxygenase) enzyme, resistance to inhibitorsof phytoene desaturase (PDS), resistance to photosystem II inhibitingherbicides (e.g., psbA), resistance to photosystem I inhibitingherbicides, resistance to protoporphyrinogen oxidase IX (PPO)-inhibitingherbicides (e.g., PPO-1), resistance to phenylurea herbicides (e.g.,(CYP76B1), dicamba-degrading enzymes (see, e.g., US 20030135879), andothers could be stacked alone or in multiple combinations to provide theability to effectively control or prevent weed shifts and/or resistanceto any herbicide of the aforementioned classes.

Additionally, soybean event 9582.814.19.1 can be combined with one ormore additional input (e.g., insect resistance, pathogen resistance, orstress tolerance, et al.) or output (e.g., increased yield, improved oilprofile, improved fiber quality, et al.) traits. Thus, the subjectinvention can be used to provide a complete agronomic package ofimproved crop quality with the ability to flexibly and cost effectivelycontrol any number of agronomic pests.

Methods to integrate a polynucleotide sequence within a specificchromosomal site of a plant cell via homologous recombination have beendescribed within the art. For instance, site specific integration asdescribed in US Patent Application Publication No. 2009/0111188 A1,herein incorporated by reference, describes the use of recombinases orintegrases to mediate the introduction of a donor polynucleotidesequence into a chromosomal target. In addition, International PatentApplication No. WO 2008/021207, herein incorporated by reference,describes zinc finger mediated-homologous recombination to integrate oneor more donor polynucleotide sequences within specific locations of thegenome. The use of recombinases such as FLP/FRT as described in U.S.Pat. No. 6,720,475, herein incorporated by reference, or CRE/LOX asdescribed in U.S. Pat. No. 5,658,772, herein incorporated by reference,can be utilized to integrate a polynucleotide sequence into a specificchromosomal site. Finally the use of meganucleases for targeting donorpolynucleotides into a specific chromosomal location was described inPuchta et al., PNAS USA 93 (1996) pp. 5055-5060).

Other methods for site specific integration within plant cells aregenerally known and applicable (Kumar et al., Trends in Plant Sci. 6(4)(2001) pp. 155-159). Furthermore, site-specific recombination systemswhich have been identified in several prokaryotic and lower eukaryoticorganisms may be applied to use in plants. Examples of such systemsinclude, but are not limited too; the R/RS recombinase system from thepSR1 plasmid of the yeast Zygosaccharomyces rouxii (Araki et al. (1985)J. Mol. Biol. 182: 191-203), and the Gin/gix system of phage Mu (Maeserand Kahlmann (1991) Mol. Gen. Genet. 230: 170-176).

In some embodiments of the present invention, it can be desirable tointegrate or stack a new transgene(s) in proximity to an existingtransgenic event. The transgenic event can be considered a preferredgenomic locus which was selected based on unique characteristics such assingle insertion site, normal Mendelian segregation and stableexpression, and a superior combination of efficacy, including herbicidetolerance and agronomic performance in and across multiple environmentallocations. The newly integrated transgenes should maintain the transgeneexpression characteristics of the existing transformants. Moreover, thedevelopment of assays for the detection and confirmation of the newlyintegrated event would be overcome as the genomic flanking sequences andchromosomal location of the newly integrated event are alreadyidentified. Finally, the integration of a new transgene into a specificchromosomal location which is linked to an existing transgene wouldexpedite the introgression of the transgenes into other geneticbackgrounds by sexual out-crossing using conventional breeding methods.

In some embodiments of the present invention, it can be desirable toexcise polynucleotide sequences from a transgenic event. For instancetransgene excision as described in Provisional U.S. Patent ApplicationNo. 61/297,628, herein incorporated by reference, describes the use ofzinc finger nucleases to remove a polynucleotide sequence, consisting ofa gene expression cassette, from a chromosomally integrated transgenicevent. The polynucleotide sequence which is removed can be a selectablemarker. Upon excision and removal of a polynucleotide sequence themodified transgenic event can be retargeted by the insertion of apolynucleotide sequence. The excision of a polynucleotide sequence andsubsequent retargeting of the modified transgenic event providesadvantages such as re-use of a selectable marker or the ability toovercome unintended changes to the plant transcriptome which resultsfrom the expression of specific genes.

The subject invention discloses herein a specific site on chromosome 02in the soybean genome that is excellent for insertion of heterologousnucleic acids. Thus, the subject invention provides methods to introduceheterologous nucleic acids of interest into this pre-established targetsite or in the vicinity of this target site. The subject invention alsoencompasses a soybean seed and/or a soybean plant comprising anyheterologous nucleotide sequence inserted at the disclosed target siteor in the general vicinity of such site. One option to accomplish suchtargeted integration is to excise and/or substitute a different insertin place of the pat expression cassette exemplified herein. In thisgeneral regard, targeted homologous recombination, for example andwithout limitation, can be used according to the subject invention.

As used herein gene, event or trait “stacking” is combining desiredtraits into one transgenic line. Plant breeders stack transgenic traitsby making crosses between parents that each have a desired trait andthen identifying offspring that have both of these desired traits.Another way to stack genes is by transferring two or more genes into thecell nucleus of a plant at the same time during transformation. Anotherway to stack genes is by re-transforming a transgenic plant with anothergene of interest. For example, gene stacking can be used to combine twoor more different traits, including for example, two or more differentinsect traits, insect resistance trait(s) and disease resistancetrait(s), two or more herbicide resistance traits, and/or insectresistance trait(s) and herbicide resistant trait(s). The use of aselectable marker in addition to a gene of interest can also beconsidered gene stacking.

“Homologous recombination” refers to a reaction between any pair ofnucleotide sequences having corresponding sites containing a similarnucleotide sequence through which the two nucleotide sequences caninteract (recombine) to form a new, recombinant DNA sequence. The sitesof similar nucleotide sequence are each referred to herein as a“homology sequence.” Generally, the frequency of homologousrecombination increases as the length of the homology sequenceincreases. Thus, while homologous recombination can occur between twonucleotide sequences that are less than identical, the recombinationfrequency (or efficiency) declines as the divergence between the twosequences increases. Recombination may be accomplished using onehomology sequence on each of the donor and target molecules, therebygenerating a “single-crossover” recombination product. Alternatively,two homology sequences may be placed on each of the target and donornucleotide sequences. Recombination between two homology sequences onthe donor with two homology sequences on the target generates a“double-crossover” recombination product. If the homology sequences onthe donor molecule flank a sequence that is to be manipulated (e.g., asequence of interest), the double-crossover recombination with thetarget molecule will result in a recombination product wherein thesequence of interest replaces a DNA sequence that was originally betweenthe homology sequences on the target molecule. The exchange of DNAsequence between the target and donor through a double-crossoverrecombination event is termed “sequence replacement.”

A preferred plant, or a seed, of the subject invention comprises in itsgenome operative cry1F v3, cry1Ac synpro and pat v6 nucleotidesequences, as identified herein, together with at least 20-500 or morecontiguous flanking nucleotides on both sides of the insert, asidentified herein. Unless indicated otherwise, reference to flankingsequences refers to those identified with respect to SEQ ID NOS: 1 and2. All or part of these flanking sequences could be expected to betransferred to progeny that receives the inserted DNA as a result of asexual cross of a parental line that includes the event.

The subject invention includes tissue cultures of regenerable cells of aplant of the subject invention. Also included is a plant regeneratedfrom such tissue culture, particularly where said plant is capable ofexpressing all the morphological and physiological properties of anexemplified variety. Preferred plants of the subject invention have allthe physiological and morphological characteristics of a plant grownfrom the deposited seed. This invention further comprises progeny ofsuch seed and seed possessing the quality traits of interest.

As used herein, a “line” is a group of plants that display little or nogenetic variation between individuals for at least one trait. Such linesmay be created by several generations of self-pollination and selection,or vegetative propagation from a single parent using tissue or cellculture techniques.

As used herein, the terms “cultivar” and “variety” are synonymous andrefer to a line which is used for commercial production.

“Stability” or “stable” means that with respect to the given component,the component is maintained from generation to generation and,preferably, at least three generations.

“Commercial Utility” is defined as having good plant vigor and highfertility, such that the crop can be produced by farmers usingconventional farming equipment, and the oil with the describedcomponents can be extracted from the seed using conventional crushingand extraction equipment

“Agronomically elite” means that a line has desirable agronomiccharacteristics such as yield, maturity, disease resistance, and thelike, in addition to the insect resistance and herbicide tolerance dueto the subject event(s). Any and all of these agronomic characteristicsand data points can be used to identify such plants, either as a pointor at either end or both ends of a range of characteristics used todefine such plants.

As one skilled in the art will recognize in light of this disclosure,preferred embodiments of detection kits, for example, can include probesand/or primers directed to and/or comprising “junction sequences” or“transition sequences” (where the soybean genomic flanking sequencemeets the insert sequence). For example, this includes a polynucleotideprobes, primers, and/or amplicons designed to identify one or bothjunction sequences (where the insert meets the flanking sequence), asindicated in the Table above. One common design is to have one primerthat hybridizes in the flanking region, and one primer that hybridizesin the insert. Such primers are often each about at least ˜15 residuesin length. With this arrangement, the primers can be used togenerate/amplify a detectable amplicon that indicates the presence of anevent of the subject invention. These primers can be used to generate anamplicon that spans (and includes) a junction sequence as indicatedabove.

The primer(s) “touching down” in the flanking sequence is typically notdesigned to hybridize beyond about 1200 bases or so beyond the junction.Thus, typical flanking primers would be designed to comprise at least 15residues of either strand within 1200 bases into the flanking sequencesfrom the beginning of the insert. That is, primers comprising a sequenceof an appropriate size from (or hybridizing to) base pairs 800 to 1400of SEQ ID NO:14 and/or base pairs 13,897 to 14,497 of SEQ ID NO:14 arewithin the scope of the subject invention. Insert primers can likewisebe designed anywhere on the, but base pairs 1400 to 2000 of SEQ ID NO:14and/or base pairs 13,297 to 13,896 of SEQ ID NO:14, and can be used, forexample, non-exclusively for such primer design.

One skilled in the art will also recognize that primers and probes canbe designed to hybridize, under a range of standard hybridization and/orPCR conditions wherein the primer or probe is not perfectlycomplementary to the exemplified sequence. That is, some degree ofmismatch can be tolerated. For an approximately 20 nucleotide primer,for example, typically one or two or so nucleotides do not need to bindwith the opposite strand if the mismatched base is internal or on theend of the primer that is opposite the amplicon. Various appropriatehybridization conditions are provided below. Synthetic nucleotideanalogs, such as inosine, can also be used in probes. Peptide nucleicacid (PNA) probes, as well as DNA and RNA probes, can also be used. Whatis important is that such probes and primers are diagnostic for (able touniquely identify and distinguish) the presence of an event of thesubject invention.

It should be noted that errors in PCR amplification can occur whichmight result in minor sequencing errors, for example. That is, unlessotherwise indicated, the sequences listed herein were determined bygenerating long amplicons from soybean genomic DNAs, and then cloningand sequencing the amplicons. It is not unusual to find slightdifferences and minor discrepancies in sequences generated anddetermined in this manner, given the many rounds of amplification thatare necessary to generate enough amplicon for sequencing from genomicDNAs. One skilled in the art should recognize and be put on notice thatany adjustments needed due to these types of common sequencing errors ordiscrepancies are within the scope of the subject invention.

It should also be noted that it is not uncommon for some genomicsequence to be deleted, for example, when a sequence is inserted duringthe creation of an event. Thus, some differences can also appear betweenthe subject flanking sequences and genomic sequences listed in GENBANK,for example.

Components of the DNA sequence “insert” are illustrated in the Figuresand are discussed in more detail below in the Examples. The DNApolynucleotide sequences of these components, or fragments thereof, canbe used as DNA primers or probes in the methods of the presentinvention.

In some embodiments of the invention, compositions and methods areprovided for detecting the presence of the transgene/genomic insertionregion, in plants and seeds and the like, from a soybean plant. DNAsequences are provided that comprise the subject 5′ transgene/genomicinsertion region junction sequence provided herein (between base pairs800 to 1400 of SEQ ID NO:14), segments thereof, and complements of theexemplified sequences and any segments thereof. DNA sequences areprovided that comprise the subject 3′ transgene/genomic insertion regionjunction sequence provided herein (between base pairs 13,897 to 14,497of SEQ ID NO:14), segments thereof, and complements of the exemplifiedsequences and any segments thereof. The insertion region junctionsequence spans the junction between heterologous DNA inserted into thegenome and the DNA from the soybean cell flanking the insertion site.Such sequences can be diagnostic for the given event.

Based on these insert and border sequences, event-specific primers canbe generated. PCR analysis demonstrated that soybean lines of thesubject invention can be identified in different soybean genotypes byanalysis of the PCR amplicons generated with these event-specific primersets. These and other related procedures can be used to uniquelyidentify these soybean lines. Thus, PCR amplicons derived from suchprimer pairs are unique and can be used to identify these soybean lines.

In some embodiments, DNA sequences that comprise a contiguous fragmentof the novel transgene/genomic insertion region are an aspect of thisinvention. Included are DNA sequences that comprise a sufficient lengthof polynucleotides of transgene insert sequence and a sufficient lengthof polynucleotides of soybean genomic sequence from one or more of thethree aforementioned soybean plants and/or sequences that are useful asprimer sequences for the production of an amplicon product diagnosticfor one or more of these soybean plants.

Related embodiments pertain to DNA sequences that comprise at least 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or morecontiguous nucleotides of a transgene portion of a DNA sequenceidentified herein (such as SEQ ID NO: I and segments thereof), orcomplements thereof, and a similar length of flanking soybean DNAsequence from these sequences, or complements thereof. Such sequencesare useful as DNA primers in DNA amplification methods. The ampliconsproduced using these primers are diagnostic for any of the soybeanevents referred to herein. Therefore, the invention also includes theamplicons produced by such DNA primers and homologous primers.

This invention also includes methods of detecting the presence of DNA,in a sample, that corresponds to the soybean event referred to herein.Such methods can comprise: (a) contacting the sample comprising DNA witha primer set that, when used in a nucleic acid amplification reactionwith DNA from at least one of these soybean events, produces an ampliconthat is diagnostic for said event(s); (b) performing a nucleic acidamplification reaction, thereby producing the amplicon; and (c)detecting the amplicon.

Further detection methods of the subject invention include a method ofdetecting the presence of a DNA, in a sample, corresponding to saidevent, wherein said method comprises: (a) contacting the samplecomprising DNA with a probe that hybridizes under stringenthybridization conditions with DNA from at least one of said soybeanevents and which does not hybridize under the stringent hybridizationconditions with a control soybean plant (non-event-of-interest DNA); (b)subjecting the sample and probe to stringent hybridization conditions;and (c) detecting hybridization of the probe to the DNA.

In still further embodiments, the subject invention includes methods ofproducing a soybean plant comprising soybean event 9582.814.19.1 of thesubject invention, wherein said method comprises the steps of: (a)sexually crossing a first parental soybean line (comprising anexpression cassettes of the present invention, which confers glufosinatetolerance to plants of said line) and a second parental soybean line(that lacks this herbicide tolerance trait) thereby producing aplurality of progeny plants; and (b) selecting a progeny plant by theuse of molecular markers. Such methods may optionally comprise thefurther step of back-crossing the progeny plant to the second parentalsoybean line to producing a true-breeding soybean plant that comprisesthe insect resistant and glufosinate tolerant trait.

According to another aspect of the invention, methods of determining thezygosity of progeny of a cross with said event is provided. Said methodscan comprise contacting a sample, comprising soybean DNA, with a primerset of the subject invention. Said primers, when used in a nucleic-acidamplification reaction with genomic DNA from at least one of saidsoybean events, produces a first amplicon that is diagnostic for atleast one of said soybean events. Such methods further compriseperforming a nucleic acid amplification reaction, thereby producing thefirst amplicon; detecting the first amplicon; and contacting the samplecomprising soybean DNA with a second primer set (said second primer set,when used in a nucleic-acid amplification reaction with genomic DNA fromsoybean plants, produces a second amplicon comprising the native soybeangenomic DNA homologous to the soybean genomic region); and performing anucleic acid amplification reaction, thereby producing the secondamplicon. The methods further comprise detecting the second amplicon,and comparing the first and second amplicons in a sample, wherein thepresence of both amplicons indicates that the sample is heterozygous forthe transgene insertion.

DNA detection kits can be developed using the compositions disclosedherein and methods well known in the art of DNA detection. The kits areuseful for identification of the subject soybean event DNA in a sampleand can be applied to methods for breeding soybean plants containingthis DNA. The kits contain DNA sequences homologous or complementary tothe amplicons, for example, disclosed herein, or to DNA sequenceshomologous or complementary to DNA contained in the transgene geneticelements of the subject events. These DNA sequences can be used in DNAamplification reactions or as probes in a DNA hybridization method. Thekits may also contain the reagents and materials necessary for theperformance of the detection method.

A “probe” is an isolated nucleic acid molecule to which is attached aconventional detectable label or reporter molecule (such as aradioactive isotope, ligand, chemiluminescent agent, or enzyme). Such aprobe is complementary to a strand of a target nucleic acid, in the caseof the present invention, to a strand of genomic DNA from one of saidsoybean events, whether from a soybean plant or from a sample thatincludes DNA from the event. Probes according to the present inventioninclude not only deoxyribonucleic or ribonucleic acids but alsopolyamides and other probe materials that bind specifically to a targetDNA sequence and can be used to detect the presence of that target DNAsequence.

“Primers” are isolated/synthesized nucleic acids that are annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, then extended alongthe target DNA strand by a polymerase, e.g., a DNA polymerase. Primerpairs of the present invention refer to their use for amplification of atarget nucleic acid sequence, e.g., by the polymerase chain reaction(PCR) or other conventional nucleic-acid amplification methods.

Probes and primers are generally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216,217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412,413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425,426,427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453,454,455,456, 457, 458, 459, 460, 461, 462,463, 464, 465, 466, 467, 468, 469,470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482,483,484, 485, 486, 487, 488, 489, 490, 491, 492, 493,494, 495, 496, 497,498, 499, 500, or 1000, or 2000, or 5000 polynucleotides or more inlength. Such probes and primers hybridize specifically to a targetsequence under high stringency hybridization conditions. Preferably,probes and primers according to the present invention have completesequence similarity with the target sequence, although probes differingfrom the target sequence and that retain the ability to hybridize totarget sequences may be designed by conventional methods.

Methods for preparing and using probes and primers are described, forexample, in

Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrooket al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989. PCR-primer pairs can be derived from a known sequence, forexample, by using computer programs intended for that purpose.

Primers and probes based on the flanking DNA and insert sequencesdisclosed herein can be used to confirm (and, if necessary, to correct)the disclosed sequences by conventional methods, e.g., by re-cloning andsequencing such sequences.

The nucleic acid probes and primers of the present invention hybridizeunder stringent conditions to a target DNA sequence. Any conventionalnucleic acid hybridization or amplification method can be used toidentify the presence of DNA from a transgenic event in a sample.Nucleic acid molecules or fragments thereof are capable of specificallyhybridizing to other nucleic acid molecules under certain circumstances.As used herein, two nucleic acid molecules are said to be capable ofspecifically hybridizing to one another if the two molecules are capableof forming an anti-parallel, double-stranded nucleic acid structure. Anucleic acid molecule is said to be the “complement” of another nucleicacid molecule if they exhibit complete complementarity. As used herein,molecules are said to exhibit “complete complementarity” when everynucleotide of one of the molecules is complementary to a nucleotide ofthe other. Two molecules are said to be “minimally complementary” ifthey can hybridize to one another with sufficient stability to permitthem to remain annealed to one another under at least conventional“low-stringency” conditions. Similarly, the molecules are said to be“complementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another underconventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., 1989. Departures fromcomplete complementarity are therefore permissible, as long as suchdepartures do not completely preclude the capacity of the molecules toform a double-stranded structure. In order for a nucleic acid moleculeto serve as a primer or probe it need only be sufficiently complementaryin sequence to be able to form a stable double-stranded structure underthe particular solvent and salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. The term “stringent conditions” is functionallydefined with regard to the hybridization of a nucleic-acid probe to atarget nucleic acid (i.e., to a particular nucleic-acid sequence ofinterest) by the specific hybridization procedure discussed in Sambrooket al., 1989, at 9.52-9.55. See also, Sambrook et al., 1989 at 9.47-9.52and 9.56-9.58. Accordingly, the nucleotide sequences of the inventionmay be used for their ability to selectively form duplex molecules withcomplementary stretches of DNA fragments.

Depending on the application envisioned, one can use varying conditionsof hybridization to achieve varying degrees of selectivity of probetowards target sequence. For applications requiring high selectivity,one will typically employ relatively stringent conditions to form thehybrids, e.g., one will select relatively low salt and/or hightemperature conditions, such as provided by about 0.02 M to about 0.15 MNaCl at temperatures of about 50° C. to about 70° C. Stringentconditions, for example, could involve washing the hybridization filterat least twice with high-stringency wash buffer (0.2×SSC, 0.1% SDS, 65°C.). Appropriate stringency conditions which promote DNA hybridization,for example, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° C. are known to those skilled inthe art. For example, the salt concentration in the wash step can beselected from a low stringency of about 2.0×SSC at 50° C. to a highstringency of about 0.2×SSC at 50° C. In addition, the temperature inthe wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or either the temperature orthe salt concentration may be held constant while the other variable ischanged. Such selective conditions tolerate little, if any, mismatchbetween the probe and the template or target strand. Detection of DNAsequences via hybridization is well-known to those of skill in the art,and the teachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 areexemplary of the methods of hybridization analyses.

In a particularly preferred embodiment, a nucleic acid of the presentinvention will specifically hybridize to one or more of the primers (oramplicons or other sequences) exemplified or suggested herein, includingcomplements and fragments thereof, under high stringency conditions. Inone aspect of the present invention, a marker nucleic acid molecule ofthe present invention has the nucleic acid sequence as set forth hereinin one of the exemplified sequences, or complements and/or fragmentsthereof.

In another aspect of the present invention, a marker nucleic acidmolecule of the present invention shares between 80% and 100% or 90% and100% sequence identity with such nucleic acid sequences. In a furtheraspect of the present invention, a marker nucleic acid molecule of thepresent invention shares between 95% and 100% sequence identity withsuch sequence. Such sequences may be used as markers in plant breedingmethods to identify the progeny of genetic crosses. The hybridization ofthe probe to the target DNA molecule can be detected by any number ofmethods known to those skilled in the art, these can include, but arenot limited to, fluorescent tags, radioactive tags, antibody based tags,and chemiluminescent tags.

Regarding the amplification of a target nucleic acid sequence (e.g., byPCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridize onlyto the target nucleic-acid sequence to which a primer having thecorresponding wild-type sequence (or its complement) would bind andpreferably to produce a unique amplification product, the amplicon.

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes under stringent hybridization conditions only to thetarget sequence in a sample comprising the target sequence.

As used herein, “amplified DNA” or “amplicon” refers to the product ofnucleic-acid amplification of a target nucleic acid sequence that ispart of a nucleic acid template. For example, to determine whether thesoybean plant resulting from a sexual cross contains transgenic eventgenomic DNA from the soybean plant of the present invention, DNAextracted from a soybean plant tissue sample may be subjected to nucleicacid amplification method using a primer pair that includes a primerderived from flanking sequence in the genome of the plant adjacent tothe insertion site of inserted heterologous DNA, and a second primerderived from the inserted heterologous DNA to produce an amplicon thatis diagnostic for the presence of the event DNA. The amplicon is of alength and has a sequence that is also diagnostic for the event. Theamplicon may range in length from the combined length of the primerpairs plus one nucleotide base pair, and/or the combined length of theprimer pairs plus about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412,413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482,483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496,497, 498, 499, or 500, 750, 1000, 1250, 1500, 1750, 2000, or morenucleotide base pairs (plus or minus any of the increments listedabove). Alternatively, a primer pair can be derived from flankingsequence on both sides of the inserted DNA so as to produce an ampliconthat includes the entire insert nucleotide sequence. A member of aprimer pair derived from the plant genomic sequence may be located adistance from the inserted DNA sequence. This distance can range fromone nucleotide base pair up to about twenty thousand nucleotide basepairs. The use of the term “amplicon” specifically excludes primerdimers that may be formed in the DNA thermal amplification reaction.

Nucleic-acid amplification can be accomplished by any of the variousnucleic-acid amplification methods known in the art, including thepolymerase chain reaction (PCR). A variety of amplification methods areknown in the art and are described, inter alia, in U.S. Pat. No.4,683,195 and U.S. Pat. No. 4,683,202. PCR amplification methods havebeen developed to amplify up to 22 kb of genomic DNA. These methods aswell as other methods known in the art of DNA amplification may be usedin the practice of the present invention. The sequence of theheterologous transgene DNA insert or flanking genomic sequence from asubject soybean event can be verified (and corrected if necessary) byamplifying such sequences from the event using primers derived from thesequences provided herein followed by standard DNA sequencing of the PCRamplicon or of the cloned DNA.

The amplicon produced by these methods may be detected by a plurality oftechniques. Agarose gel electrophoresis and staining with ethidiumbromide is a common well known method of detecting DNA amplicons.Another such method is Genetic Bit Analysis where an DNA oligonucleotideis designed which overlaps both the adjacent flanking genomic DNAsequence and the inserted DNA sequence. The oligonucleotide isimmobilized in wells of a microwell plate. Following PCR of the regionof interest (using one primer in the inserted sequence and one in theadjacent flanking genomic sequence), a single-stranded PCR product canbe hybridized to the immobilized oligonucleotide and serve as a templatefor a single base extension reaction using a DNA polymerase and labeledddNTPs specific for the expected next base. Readout may be fluorescentor ELISA-based. A signal indicates presence of the insert/flankingsequence due to successful amplification, hybridization, and single baseextension.

Another method is the Pyrosequencing technique as described by Winge(Innov. Pharma. Tech. 00:18-24, 2000). In this method an oligonucleotideis designed that overlaps the adjacent genomic DNA and insert DNAjunction. The oligonucleotide is hybridized to single-stranded PCRproduct from the region of interest (one primer in the inserted sequenceand one in the flanking genomic sequence) and incubated in the presenceof a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5′phosphosulfate and luciferin. DNTPs are added individually and theincorporation results in a light signal that is measured. A light signalindicates the presence of the transgene insert/flanking sequence due tosuccessful amplification, hybridization, and single or multi-baseextension.

Fluorescence Polarization is another method that can be used to detectan amplicon of the present invention. Following this method, anoligonucleotide is designed which overlaps the genomic flanking andinserted DNA junction. The oligonucleotide is hybridized tosingle-stranded PCR product from the region of interest (one primer inthe inserted DNA and one in the flanking genomic DNA sequence) andincubated in the presence of a DNA polymerase and a fluorescent-labeledddNTP. Single base extension results in incorporation of the ddNTP.Incorporation can be measured as a change in polarization using afluorometer. A change in polarization indicates the presence of thetransgene insert/flanking sequence due to successful amplification,hybridization, and single base extension.

TAQMAN® (PE Applied Biosystems, Foster City, Calif.) is a method ofdetecting and quantifying the presence of a DNA sequence. Briefly, aFRET oligonucleotide probe is designed that overlaps the genomicflanking and insert DNA junction. The FRET probe and PCR primers (oneprimer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. During specific amplification, Taq DNA polymerase cleans andreleases the fluorescent moiety away from the quenching moiety on theFRET probe. A fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection.Briefly, a FRET oligonucleotide probe is designed that overlaps theflanking genomic and insert DNA junction. The unique structure of theFRET probe results in it containing secondary structure that keeps thefluorescent and quenching moieties in close proximity. The FRET probeand PCR primers (one primer in the insert DNA sequence and one in theflanking genomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Following successful PCR amplification,hybridization of the FRET probe to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties. A fluorescent signal results. Afluorescent signal indicates the presence of the flankinggenomic/transgene insert sequence due to successful amplification andhybridization.

Having disclosed a location in the soybean genome that is excellent foran insertion, the subject invention also comprises a soybean seed and/ora soybean plant comprising at least one non-soybean event 9582.814.19.1insert in the general vicinity of this genomic location. One option isto substitute a different insert in place of the one from soybean eventpDAB9582.814.19.1 exemplified herein. In these general regards, targetedhomologous recombination, for example, can be used according to thesubject invention. This type of technology is the subject of, forexample, WO 03/080809 A2 and the corresponding published U.S.application (US 20030232410). Thus, the subject invention includesplants and plant cells comprising a heterologous insert (in place of orwith multi-copies of the cry1F cry1Ac, or pat genes), flanked by all ora recognizable part of the flanking sequences identified herein (bp1-1400 of SEQ ID NO:1 and bp 153-1550 of SEQ ID NO:2). An additionalcopy (or additional copies) of a cry1F, cry1Ac, or pat could also betargeted for insertion in this/these manner(s).

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification

The following examples are included to illustrate procedures forpracticing the invention and to demonstrate certain preferredembodiments of the invention. These examples should not be construed aslimiting. It should be appreciated by those of skill in the art that thetechniques disclosed in the following examples represent specificapproaches used to illustrate preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in these specific embodimentswhile still obtaining like or similar results without departing from thespirit and scope of the invention. Unless otherwise indicated, allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

The following abbreviations are used unless otherwise indicated.

-   -   bp base pair    -   ° C. degrees Celsius    -   DNA deoxyribonucleic acid    -   EDTA ethylenediaminetetraacetic acid    -   kb kilobase    -   μg microgram    -   μL microliter    -   mL milliliter    -   M molar mass    -   PCR polymerase chain reaction    -   PTU plant transcription unit    -   SDS sodium dodecyl sulfate    -   SSC a buffer solution containing a mixture of sodium chloride        and sodium citrate, pH 7.0    -   TBE a buffer solution containing a mixture of Tris base, boric        acid and EDTA, pH 8.3

Embodiments of the present invention are further defined in thefollowing Examples. It should be understood that these Examples aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theembodiments of the invention to adapt it to various usages andconditions. Thus, various modifications of the embodiments of theinvention, in addition to those shown and described herein, will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

The disclosure of each reference set forth herein is incorporated hereinby reference in its entirety.

EXAMPLES Example 1 Transformation and Selection of the Cry1F and Cry1AcSoybean Event pDAB9582.814.19.1

Transgenic soybean (Glycine max) containing the soybean eventpDAB9582.814.19.1 was generated through Agrobacterium-mediatedtransformation of soybean cotyledonary node explants. The disarmedAgrobacterium strain EHA101 (Hood et al., 1993), carrying the binaryvector pDAB9582 (FIG. 1) containing the selectable marker, pat v6, andthe genes of interest, cry1F v3 and cry1Ac synpro, within the T-strandDNA region, was used to initiate transformation. The DNA sequence forpDAB9582 is given in SEQ ID NO:3, which is annotated below in Table 1.

TABLE 1 Gene elements located on pDAB9582. bp (SEQ ID Construct NO: 3)element Reference  272-1593 AtUbi10 Callis, et al., (1990) J. Biol.Chem., Promoter 265: 12486-12493 1602-5048 Cry1F Referenced above5151-5607 ORF23 3′UTR U.S. Pat. No. 5,428,147 5671-6187 CsVMV Verdagueret al., (1996) Plant Mol. Promoter Biol., 31: 1129-1139 6197-9667 Cry1AC Referenced above  9701-10157 ORF23 3′UTR U.S. Pat. No. 5,428,14710272-10788 CsVMV Verdaguer et al., (1996) Plant Mol. Promoter Biol.,31: 1129-1139 10796-11347 PAT Wohlleben et al., (1988) Gene 70: 25-3711450-12153 ORF1 3′UTR Huang et al., (1990) J. Bacteriol. 172: 1814-1822

Agrobacterium-mediated transformation was carried out using a modifiedprocedure of Zeng et al. (2004). Briefly, soybean seeds (cv Maverick)were germinated on basal media and cotyledonary nodes were isolated andinfected with Agrobacterium. Shoot initiation, shoot elongation, androoting media were supplemented with cefotaxime, timentin and vancomycinfor removal of Agrobacterium. Glufosinate selection was employed toinhibit the growth of non-transformed shoots. Selected shoots weretransferred to rooting medium for root development and then transferredto soil mix for acclimatization of plantlets.

Terminal leaflets of selected plantlets were leaf painted withglufosinate to screen for putative transformants. The screened plantletswere transferred to the greenhouse, allowed to acclimate and thenleaf-painted with glufosinate to reconfirm tolerance and deemed to beputative transformants. The screened plants were sampled and molecularanalyses for the confirmation of the selectable marker gene and/or thegene of interest were carried out. T₀ plants were allowed to selffertilize in the greenhouse to give rise to T₁ seed.

This event, soybean event pDAB9582.814.19.1, was generated from anindependent transformed isolate. The T₁ plants were backcrossed andintrogressed into elite varieties over subsequent generations. The eventwas selected based on its unique characteristics such as singleinsertion site, normal Mendelian segregation, stable expression, and asuperior combination of efficacy, including herbicide tolerance andagronomic performance. The following examples contain the data whichwere used to characterize soybean event pDAB9582.814.19.1.

Example 2 Characterization of Protein Expression in Soybean EventpDAB9582.814.19.1

The biochemical properties of the recombinant Cry1F, Cry1Ac, and PATproteins expressed in soybean event 9582.814.19.1 were characterized.Quantitative enzyme-linked immunosorbent assay (ELISA) is a biochemicalassay known within the art that can be used to characterize thebiochemical properties of the proteins and confirm expression of theseproteins in soybean event 9582.814.19.1.

Example 2.1 Expression of the PAT, Cry1F, and Cry1Ac Protein in PlantTissues

Samples of soybean tissues were isolated from the test plants andprepared for expression analysis. The PAT protein was extracted fromsoybean plant tissues with a phosphate buffered saline solutioncontaining the detergent Tween-20 (PBST) containing 0.5% Bovine SerumAlbumin (BSA). The plant tissue was centrifuged; the aqueous supernatantwas collected, diluted with appropriate buffer as necessary, andanalyzed using an PAT ELISA kit in a sandwich format. The kit was usedfollowing the manufacturer's suggested protocol (Envirologix, Portland,Me.). This assay measured the expressed PAT protein.

The Cry1F protein was extracted from soybean plant tissues with aphosphate buffered saline solution containing the detergent Tween-20(PBST). The plant tissue was centrifuged; the aqueous supernatant wascollected, diluted with appropriate buffer as necessary, and analyzedusing an Cry1F ELISA kit in a sandwich format. The kit was usedfollowing the manufacturer's suggested protocol (Strategic DiagnosticsInc., Newark, Del.). This assay measured the expressed Cry1F protein.

The Cry1Ac protein was extracted from soybean plant tissues with aphosphate buffered saline solution containing the detergent Tween-20(PBST) containing 0.5% Bovine Serum Albumin (BSA). The plant tissue wascentrifuged; the aqueous supernatant was collected, diluted withappropriate buffer as necessary, and analyzed using an Cry1Ac ELISA kitin a sandwich format. The kit was used following the manufacturer'ssuggested protocol (Strategic Diagnostics Inc., Newark, Del.). Thisassay measured the Cry1Ac protein.

Detection analysis was performed to investigate the expression stabilityand inheritability both vertically (between generations) andhorizontally (between lineages within a generation) in soybean eventpDAB9582.814.19.1.

Example 2.2 Expression of the PAT, Cry1F, and Cry1Ac Protein in PlantTissues

Levels of Cry1F, Cry1Ac and PAT proteins were determined in SoybeanEvent 9582.814.19.1. The soluble, extractable proteins were measuredusing a quantitative enzyme-linked immunosorbent assay (ELISA) methodfrom soybean leaf tissue. From T₂ to T₆ generations Soybean Events9582.814.19.1, expression was stable (not segregating) and consistentacross all lineages. Table 2 lists the mean expression level of thetransgenic proteins in soybean event 9582.814.19.1.

TABLE 2 Mean expression level of different transgenic proteins insoybean event pDAB9582.814.19.1. Expression Level of Different Proteins(ng/cm²) Event Cry1F Cry1 Ac PAT Soybean event 133 17.4 12pDAB9582.814.19.1

Example 3 Cloning and Characterization of DNA Sequence in the Insert andthe Flanking Border Regions of Soybean Event pDAB9582.814.1.9.1

To characterize and describe the genomic insertion site, the sequence ofthe flanking genomic T-DNA border regions of soybean eventpDAB9582.814.19.1 were determined. Genomic sequence of soybean eventpDAB9582.814.19.1 was confirmed, comprising 1400 bp of 5′ flankingborder sequence (SEQ ID NO:1) and 1398 bp of 3′ flanking border sequence(SEQ ID NO:2). PCR amplification based on the soybean eventpDAB9582.814.19.1 border sequences validated that the border regionswere of soybean origin and that the junction regions are uniquesequences for soybean event pDAB9582.814.19.1. The junction regionscould be used for event-specific identification of soybean eventpDAB9582.814.19.1. In addition, the T-strand insertion site wascharacterized by amplifying a genomic fragment corresponding to theregion of the identified flanking border sequences from the genome ofuntransformed soybean. Comparison of soybean event pDAB9582.814.19.1with the untransformed genomic sequence revealed that a deletion ofabout 57 bp from the original locus resulted during the T-strandintegration. Overall, the characterization of the insert and bordersequence of soybean event pDAB9582.814.19.1 indicated that an intactcopy of the T-strand from pDAB9582 was present in the soybean genome.

TABLE 3 List of primers and their sequences used in the confirmation  of soybean genomic DNA in soybean event pDAB9582.814.19.1 SEQ ID PrimerSize NO: Name (bp) Sequence (5′ to 3′) Purpose SEQ ID 81419_FW3 30TTTCTCCTATCCGTC confirmation of 5′ border NO: 4 AAATAAATCTGCTCCgenomic DNA, used with AtUbi10RV1 or RV2; with 5′ IREnd-01 or 5′IREnd-02SEQ ID 81419_RV1 27 GGGTGATTTGGTGCC confirmation of 3′ border NO: 5AAAAGTTATGTT genomic DNA, used with 3′PATEnd05 or 3′PATEnd06 SEQ ID81419_RV2 24 TGGAGGGTCATATCG confirmation of 3′ border NO: 6 CAAAAGACTgenomic DNA, used with 3′PATEnd05 or 3′PATEnd06 SEQ ID  81419_RV3 24GTTCTGCGTCGTGGA confirmation of 3′ border NO: 7 GGGTCATATgenomic DNA, used with 3′PATEnd05 or 3′PATEnd06 SEQ ID 5′IREnd-01 29CGAGCTTTCTAATTT confirmation of 5′ border NO: 8 CAAACTATTTCGGGCgenomic DNA, used with 81419_FW3 SEQ ID 5′IREnd-02 30 TCCTAGATCATCAGTconfirmation of 5′ border NO: 9 TCATACAAACCTCCA  genomiC DNA, used with81419_FW3 SEQ ID AtUbi10 29 CGGTCCTAGATCATC  confirmation of 5′ borderNO: 10 RV1 AGTTCATACAAACC genomic DNA, used with 81419_FW3 SEQ IDAtUbil0 28 CACTCGTGTTCAGTC confirmation of 5′ border NO: 11 RV2CAATGACCAATAA genomic DNA, used with 81419_FW3 SEQ ID 3′PATE 20GCTCCTCCAAGGCCA confirmation of 3′ border NO: 12 nd05 GTTAGgenomic DNA, used with 81419_RV1, RV2 or RV3 SEQ ID 3′PATE 20CCAGTTAGGCCAGTT confirmation of 3′ border NO: 13 nd06 ACCCAgenomic DNA, used with 81419_RV1, RV2 or RV3

TABLE 4 Conditions for standard PCR amplification of the border regionsand event-specific sequences in soybean event pDAB9582.814.19.1. Pre-Final Target PCR denature Denature Extension Extension Sequence PrimerSet Mixture (° C./min) (° C./sec.) (° C./min:sec) (° C./min) 5′ border81419_FW3/ D 95/3 98/10 68/4:00 72/10 AtUbi10RV1 32 cycles 5′ border81419_FW3/ D 95/3 98/10 68/4:00 72/10 5′IREnd- 32 cycles 01 3′ border3′PATEnd05/ D 95/3 98/10 68/4:00 72/10 81419_RV2 35 cycles 3′ border3′PATEnd05/ D 95/3 98/10 68/4:00 72/10 81419_RV3 35 cycles 3′ border3′PATEnd06/ D 95/3 98/10 68/4:00 72/10 81419_RV2 35 cycles 3′border3′PATEnd06/ D 95/3 98/10 68/4:00 72/10 81419_RV3 32 cycles Across81419_FW3/ D 95/3 98/10 68/4:00 72/10 the insert 81419_RV3 32 cycleslocus

TABLE 5 PCR mixture for standard PCR amplification of the border regionsand event specific sequences in soybean event pDAB9582.814.19.1. 1 xreaction 1 x reaction Reagent (μL) Reagent (μL) PCR Mixture A PCRMixture B H20 0.8 H20 14.6 ACCPRIME PFX 20 10X LA TAQ 2 SUPERMIX BUFFER— — MgCl2 (25 mM) 0.6 — — dNTP (2.5 uM) 1.6 10 uM primer 0.2 10 uMprimer 0.1 gDNA digestion 1 gDNA digestion 1 — — LA TAQ (5U/ul) 0.1 rxnvol: 22 rxn vol: 20 PCR Mixture C PCR Mixture D H20 28 H20 11.6 10X PCRbuffer II 5 10X PCR buffer II 2 (Mg-plus) (Mg-plus) MgCl₂ [25 mM] 1.5MgCl₂ [25 mM] 0.6 dNTP [2.5 mM] 8 dNTP [2.5 mM] 3.2 Adaptor PCR primer 1primer1 (10 μM) 0.4 (10 μM) GOI nested primer 1 primer2 (10 μM) 0.4 (10μM) DNA binded Beads 5 DNA Template 0.2 LA TAQ (5U/ul) 0.5 LA TAQ(5U/ul) 1.6 rxn vol: 50 rxn vol: 20

Example 3.1 Confirmation of Soybean Genomic Sequences

The 5′ and 3′ flanking borders aligned to a Glycine max whole genomeshotgun sequence from chromosome 02, indicating that the transgene ofsoybean event pDAB9582.814.19.1 was inserted in soybean genomechromosome 02. To confirm the insertion site of soybean eventpDAB9582.814.19.1 from the soybean genome, PCR was carried out withdifferent pairs of primers (FIG. 2, Table 3, Table 4, and Table 5).Genomic DNA from soybean event pDAB9582.814.19.1 and other transgenic ornon-transgenic soybean lines was used as a template. To confirm that the5′ border sequences are correct a primer designed to bind to the AtUbi10 promoter gene element, for example AtUbi10RV1, and a primerdesigned to bind to the cloned 5′ end border on soybean genomechromosome 02, primer designated 81419_FW3, were used for amplifying theDNA segment that spans the At Ubi10 promoter gene element to 5′ endborder sequence. Similarly, for confirmation of the cloned 3′ bordersequence a pat specific primer, for example 3′PATEnd05, and threeprimers designed according to the cloned 3′ end border sequence,designated 81419_RV1, 81419_RV2 and 81419_RV3, were used for amplifyingDNA segments that span the pal gene to 3′ border sequence. DNA fragmentswith expected sizes were amplified only from the genomic DNA of soybeanevent pDAB9582.814.19.1 with each primer pair, but not from DNA samplesfrom other transgenic soybean lines or the non-transgenic control. Theresults indicate that the cloned 5′ and 3′ border sequences are theflanking border sequences of the T-strand insert for soybean eventpDAB9582.814.19.1.

To further confirm the DNA insertion in the soybean genome, a PCRamplification spanning the soybean border sequences was completed ongenomic DNA which did not contain the T-strand insert for soybean eventpDAB9582.814.19.1. Primer 81419_FW3, designed according to the 5′ endborder sequence, and one primer 81419-RV3, designed for the 3′ endborder sequence, were used to amplify DNA segments which contained thelocus where the pDAB9582 T-strand integrated. As expected, PCRamplification completed with the primer pair of 81419_FW3 and 81419_RV3produced an approximately a 1.5 kb DNA fragment from all the othersoybean control lines but not pDAB9582.814.19.1. Aligning the identified5′ and 3′ border sequences of soybean event pDAB9582.814.19.1 with aGlycine max whole genome shotgun sequence from chromosome 02 revealedabout 57 bp deletion from the original locus. (FIG. 3). These resultsdemonstrated that the transgene of soybean event pDAB8294 was insertedinto the site of soybean genome chromosome 02.

Example 4 Soybean Event pDAB9582.814.19.1 Characterization via SouthernBlot

Southern blot analysis was used to establish the integration pattern ofsoybean event pDAB9582.814.19.1. These experiments generated data whichdemonstrated the integration and integrity of the cry1Ac and cry1Ftransgenes within the soybean genome. Soybean event pDAB9582.814.19.1was characterized as a full length, simple integration event containinga single copy of the cry1Ac and cry1F plant transcription unit (PTU)from plasmid pDAB9582.

Southern blot data suggested that a T-strand fragment inserted into thegenome of soybean event pDAB9582.814.19.1. Detailed Southern blotanalysis was conducted using probes specific to the cry1Ac and cry1Fgene, contained in the T-strand integration region of pDAB9582.814.19.1,and descriptive restriction enzymes that have cleavage sites locatedwithin the plasmid and produce hybridizing fragments internal to theplasmid or fragments that span the junction of the plasmid with soybeangenomic DNA (border fragments). The molecular weights indicated from theSouthern hybridization for the combination of the restriction enzyme andthe probe were unique for the event, and established its identificationpatterns. These analyses also showed that the plasmid fragment had beeninserted into soybean genomic DNA without rearrangements of the cry1Acand cry1F PTU.

Example 4.1 Soybean Leaf Sample Collection and Genomic DNA (gDNA)Isolation

Genomic DNA was extracted from leaf tissue harvested from individualsoybean plants containing soybean event pDAB9582.814.19.1. In addition,gDNA was isolated from a conventional soybean plant, Maverick, whichcontains the genetic background that is representative of the substanceline, absent the cry1Ac and cry1F genes. Individual genomic DNA wasextracted from lyophilized leaf tissue following the standard CTABmethod (Sambrook et al (1989)). Following extraction, the DNA wasquantified spectrofluorometrically using PICO GREEN reagent (Invitrogen,Carlsbad, Calif.). The DNA was then visualized on an agarose gel toconfirm values from the PICO GREEN analysis and to determine the DNAquality.

Example 4.2 DNA Digestion and Separation

For Southern blot molecular characterization of soybean eventpDAB9582.814.19.1, ten micrograms (10 μg) of genomic DNA was digested.Genomic DNA from the soybean event pDAB9582.814.19.1 and non-transgenicsoybean line Maverick was digested by adding approximately five units ofselected restriction enzyme per μg of DNA and the corresponding reactionbuffer to each DNA sample. Each sample was incubated at approximately37° C. overnight. The restriction enzymes AseI, HindIII, NsiI, and NdeIwere used individually for the single digests (New England Biolabs,Ipswich, Mass.). The restriction enzymes NotI and ApaLI were usedtogether for a double digestion (New England Biolabs, Ipswich, Mass.).In addition, a positive hybridization control sample was prepared bycombining plasmid DNA, pDAB9582 with genomic DNA from the non-transgenicsoybean variety, Maverick. The plasmid DNA/genomic DNA cocktail wasdigested using the same procedures and restriction enzyme as the testsamples.

After the digestions were incubated overnight, 25 μL QUICK-PRECIP PLUSSOLUTION (Edge Biosystems, Gaithersburg, Md.) was added and the digestedDNA samples were precipitated with isopropanol. The precipitated DNApellet was resuspended in 15 μL of 1× loading buffer (0.01% bromophenolblue, 10.0 mM EDTA, 10.0% glycerol, 1.0 mM Tris pH 7.5). The DNA samplesand molecular size markers were then electrophoresed through 0.85%agarose gels with 0.4×TAE buffer (Fisher Scientific, Pittsburgh, Pa.) at35 volts for approximately 18-22 hours to achieve fragment separation.The gels were stained with ethidium bromide (Invitrogen, Carlsbad,Calif.) and the DNA was visualized under ultraviolet (UV) light.

Example 4.3 Southern Transfer and Membrane Treatment

Southern blot analysis was performed essentially as described byMemelink, et al. (1994). Briefly, following electrophoretic separationand visualization of the DNA fragments, the gels were depurinated with0.25M HCl for approximately 20 minutes, and then exposed to a denaturingsolution (0.4 M NaOH, 1.5 M NaCl) for approximately 30 minutes followedby neutralizing solution (1.5 M NaCl, 0.5 M Tris pH 7.5) for at least 30minutes. Southern transfer was performed overnight onto nylon membranesusing a wicking system with 10×SSC. After transfer the DNA was bound tothe membrane by UV crosslinking following by briefly washing membranewith a 2×SSC solution. This process produced Southern blot membranesready for hybridization.

Example 4.4 DNA Probe Labeling and Hybridization

The DNA fragments bound to the nylon membrane were detected using alabeled probe (Table 6). Probes were generated by a PCR-basedincorporation of a digoxigenin (DIG) labeled nucleotide, [DIG-11]-dUTP,into the DNA fragment amplified from plasmid pDAB9582 using primersspecific to gene elements. Generation of DNA probes by PCR synthesis wascarried out using a PCR DIG Probe Synthesis Kit (Roche Diagnostics,Indianapolis, Ind.) following the manufacturer's recommended procedures.

Labeled probes were analyzed by agarose gel electrophoresis to determinetheir quality and quantity. A desired amount of labeled probe was thenused for hybridization to the target DNA on the nylon membranes fordetection of the specific fragments using the procedures essentially asdescribed for DIG EASY HYB SOLUTION (Roche Diagnostics, Indianapolis,Ind.). Briefly, nylon membrane blots containing fixed DNA were brieflywashed with 2×SSC and pre-hybridized with 20-25 mL of pre-warmed DIGEASY HYB SOLUTION in hybridization bottles at approximately 45-55° C.for about 2 hours in a hybridization oven. The pre-hybridizationsolution was then decanted and replaced with ˜15 mL of pre-warmed DIGEASY HYB SOLUTION containing a desired amount of specific probesdenatured by boiling in a water bath for approximately five minutes. Thehybridization step was then conducted at approximately 45-55° C.overnight in the hybridization oven.

At the end of the probe hybridization, DIG EASY HYB SOLUTIONS containingthe probes were decanted into clean tubes and stored at approximately−20° C. These probes could be reused up to two times according to themanufacturer's recommended procedure. The membrane blots were rinsedbriefly and washed twice in clean plastic containers with low stringencywash buffer (2×SSC, 0.1% SDS) for approximately five minutes at roomtemperature, followed by washing twice with high stringency wash buffer(0.1×SSC, 0.1% SDS) for 15 minutes each at approximately 65° C. Themembrane blots briefly washed with 1× Maleic acid buffer from the DIGWASH AND BLOCK BUFFER SET (Roche Diagnostics, Indianapolis, Ind.) forapproximately 5 minutes. This was followed by blocking in a IX blockingbuffer for 2 hours and an incubation with anti-DIG-AP (alkalinephosphatase) antibody (Roche Diagnostics, Indianapolis, Ind.) in 1×blocking buffer also for a minimum of 30 minutes. After 2-3 washes with1× washing buffer, specific DNA probes remain bound to the membraneblots and DIG-labeled DNA standards were visualized using CDP-STARCHEMILUMINESCENT NUCLEIC ACID DETECTION SYSTEM (Roche Diagnostics,Indianapolis, Ind.) following the manufacturer's recommendation. Blotswere exposed to chemiluminescent film for one or more time points todetect hybridizing fragments and to visualize molecular size standards.Films were developed with an ALL-PRO 100 PLUS film developer (KonicaMinolta, Osaka, Japan) and images were scanned. The number and sizes ofdetected bands were documented for each probe. DIG-LABELED DNA MOLECULARWEIGHT MARKER II (DIG MWM II) and DIG-LABELED DNA MOLECULAR WEIGHTMARKER VII (DIG MWM VII), visible after DIG detection as described, wereused to determine hybridizing fragment size on the Southern blots.

TABLE 6 Location and length of probes used in Southern analysis. ProbeName Genetic Element Length (bp) Cry1Ac cry1Ac 1720 Cry1F cry1F 1746specR Spectinomycin resistance gene 750 OriRep Ori Rep 852 trfAReplication initiation protein trfA 1119

Example 4.5 Southern Blot Results

Expected and observed fragment sizes with a particular digest and probe,based on the known restriction enzyme sites of the cry1Ac and cry1F PTU,are given in Table 7. Two types of fragments were identified from thesedigests and hybridizations: internal fragments where known enzyme sitesflank the probe region and are completely contained within the insertionregion of the cry1Ac and cry1F PTU, and border fragments where a knownenzyme site is located at one end of the probe region and a second siteis expected in the soybean genome. Border fragment sizes vary by eventbecause, in most cases, DNA fragment integration sites are unique foreach event. The border fragments provide a means to locate a restrictionenzyme site relative to the integrated DNA and to evaluate the number ofDNA insertions. Southern blot analyses completed on multiple generationsof soybean containing soybean event pDAB9582.814.19.1 produced datawhich suggested that a low copy, intact cry1Ac and cry1F PTU fromplasmid pDAB9582 was inserted into the soybean genome of soybean eventpDAB9582.814.19.1.

TABLE 7 Predicted and observed hybridizing fragments in Southern blotanalysis. 1. Expected fragment sizes are based on the plasmid map ofpDAB9582. 2. Observed fragment sizes are considered approximately fromthese analyses and are based on the indicated sizes of the DIG-LABELEDDNA MOLECULAR WEIGHT MARKER II and MARK VII fragments. Expected ObservedDNA Restriction Fragment Fragment Probe Enzymes Samples Sizes (bp)¹ Size(bp)² Cry1Ac AseI pDAB9582 13476 >14000 Maverick none none SoybeanEvent >7286 ~7400 pDAB9582.814.19.1 Nsi I pDAB9582 15326 >15000 Mavericknone none Soybean Event >9479 >10000 pDAB9582.814.19.1 Not I + pDAB95824550 ~4500 ApaLI Maverick none none Soybean Event 4550 ~4500pDAB9582.814.19.1 Cry1F NdeI pDAB9582 8071 ~8000 Maverick none noneSoybean Event 5569 ~7500 pDAB9582.814.19.1 Nsi I pDAB9582 11044 11000Maverick none none Soybean Event >9479 >10000 pDAB9582.814.19.1 Hind IIIpDAB9582 7732 ~7700 Maverick none none Soybean Event 7732 ~7700pDAB9582.814.19.1 SpecR NsiI pDAB9582 15320 ~15000 Maverick none noneSoybean Event none none pDAB9582.814.19.1 trfA NsiI pDAB9582 15320~15000 Maverick none none Soybean Event none none pDAB9582.814.19.1oriREP NdeI pDAB9582 5239 ~5000 Maverick none none Soybean Event nonenone pDAB9582.814.19.1

The restriction enzymes AseI and NsiI bind and cleave unique restrictionsites in plasmid pDAB9582. Subsequently, these enzymes were selected tocharacterize the cry1Ac gene insert in soybean event pDAB9582.814.19.1.Border fragments of >7286 bp or >9479 bp were predicted to hybridizewith the probe following AseI and NsiI digests, respectively (Table 7).Single cry1Ac hybridization bands of about 7400 and >10000 bp wereobserved when AseI and NsiI digests were used, respectively. Thehybridization of the probe to bands of this size suggests the presenceof a single site of insertion for the cry1Ac gene in the soybean genomeof soybean event pDAB9582.814.19.1. Restriction enzymes NotI and ApaLIwere selected to perform a double digestion and to release a fragmentwhich contains the cry1Ac plant transcription unit (PTU;promoter/gene/terminator) (Table 7). The predicted 4550 bp fragmentswere observed with the probe following NotI and ApaLI double digestion.Results obtained with the enzyme digestion of the pDAB9582.814.19.1samples followed by probe hybridization indicated that an intact cry1AcPTU from plasmid pDAB9582 was inserted into the soybean genome ofsoybean event pDAB9582.814.19.1.

The restriction enzymes NdeI and NsiI bind and cleave restriction sitesin plasmid pDAB9582. Subsequently, these enzymes were selected tocharacterize the cry1F gene insert in soybean event pDAB9582.814.19.1.Border fragments of >5569 bp and >9479 were predicted to hybridize withthe probe following the NdeI and NsiI digests, respectively (Table 7).Single cry1F hybridization bands of ˜7500 bp and >10000 bp were observedwhen NdeI and NsiI were used, respectively. The hybridization of theprobe to bands of this size suggests the presence of a single site ofinsertion for the cry/F gene in the soybean genome of soybean eventpDAB9582.814.19.1. Restriction enzyme, HindIII, was selected to releasea fragment which contains the cry1F plant transcription unit (PTU;promoter/gene/terminator) (Table 7). The predicted 7732 bp fragment wasobserved with the probe following the HindIII digestions. Resultsobtained with the enzyme digestion of the pDAB9582.814.19.1 samplesfollowed by probe hybridization indicated that an intact cry1F PTU fromplasmid pDAB9582 was inserted into the soybean genome of soybean eventpDAB9582.814.19.1.

Example 4.6 Absence of Backbone Sequences

Southern blot analysis was also conducted to verify the absence of thespectinomycin resistance gene (specR), Ori Rep element and replicationinitiation protein trfA (trf A element) in soybean eventpDAB9582.814.19.1. No specific hybridization to spectinomycinresistance, Ori Rep element or trf A element is expected whenappropriate positive (pDAB9582 added to Maverick genomic DNA) andnegative (Maverick genomic DNA) controls are included for Southernanalysis. Following the NsiI digestion and hybridization with the specRspecific probe, one expected size band of 15320 bp was observed in thepositive control sample (pDAB9582 added to Maverick genomic DNA). ThespecR probe did not hybridize to samples of the negative control andsoybean event pDAB9582.814.19.1. Similarly, one expected size band of15320 bp was detected in the positive control sample (pDAB9582 plusmaverick) but absent from the samples of the negative control andsoybean event pDAB9582.814.19.1 after NsiI digestion and hybridizationwith trfA probe. Another expected size band of 5329 bp was detected inthe positive control sample (pDAB9582 added to Maverick genomic DNA) butabsent from the samples of the negative control and soybean eventpDAB9582.814.19.1 after NdeI digestion and hybridization with OriRepspecific probe. These data indicate the absence of spectinomycinresistance gene, Ori Rep element and replication initiation protein trfAin soybean event pDAB9582.814.19.1.

Example 5 Agronomic and Yield Field Trial and Herbicide Tolerance

To test the agronomic characteristics and efficacy of soybean eventpDAB9582.814.19.1 the event was planted in an efficacy trial at SantaIsabel, Puerto Rico in October 2010 and February 2011. The cultivarMaverick, which was originally transformed to produce eventpDAB9582.814.19.1, was planted in each nursery and included as a controlin the experiments. Seed for the T3 nursery was derived from singleplant selections at the T2 stage and seed for the T4 nursery was derivedfrom single plant selections at the T3 stage. Four lineages of the eventwere tested each generation. Each lineage was planted in a plot whichwas 4 rows wide and 7.5 feet long. The spacing between rows was 30inches. Plots were grown under lights for approximately 2.5 weeks tocompensate for the short day length in Puerto Rico. Each nursery wassprayed with glufosinate at a rate of 411 g ae/ha. One plot of thecontrol plants, Maverick, was sprayed with the same rate of glufosinateand a second plot was non-sprayed and used as control comparison for theevent.

Data was collected on emergence, general appearance, vigor, height,lodging, and maturity. Herbicide tolerance was assessed by visuallylooking for chlorosis, leaf necrosis and plant death (Table 8).

For comparisons of soybean event pDAB9582.814.19.1 with Maverick, onlydata from the unsprayed block of Maverick were used. For comparison ofthe sprayed and non-sprayed treatments, data from the soybean eventpDAB9582.814.19.1 block sprayed with a given treatment were comparedwith data from the Maverick control non-sprayed block. Soybean eventpDAB9582.814.19.1 showed tolerance to the glufosinate herbicideapplication. In contrast, none of the Maverick plants were tolerant tothe herbicide treatments.

TABLE 8 Comparison of soybean event pDAB9582.814.19.1 to Maverick.Values are averages from T₃ and T₄ nurseries. Each nursery of soybeanevent pDAB9582.814.19.1 was sprayed with glufosinate at the V3 stage ata rate of 411 g ae/ha. Appearance Vigor Emergence (1 = poor to (1 = poorto Height Lodging Maturity Event (%) 9 = good) 9 = good) (cm) (%) (day)pDAB9582.814.19.1 90 8 8 69 1 91 Maverick 82 8 8 64 1 91

Example 6 Characterization of Insecticidal Activity for Soybean Event9582.814.19.1

Field and greenhouse evaluations were conducted to characterize theactivity of Cry1Ac and Cry1F in soybean event pDA B9582.814.19.1 againstlab reared soybean pests including Anticarsia gemmatalis (velvetbeancaterpillar), Pseudoplusia includens (soybean looper) and Spodopterafrugiperda (fall armyworm). Soybean event pDAB9582.814.19.1 was comparedagainst non-transformed soybean variety Maverick, to determine the levelof plant protection provided by the Cry1F and Cry1Ac proteins.

Greenhouse trials were conducted on approximately four week old plants.Fifteen plants were used to evaluate the soybean event pDAB9582.814.19.1and the Maverick control. For each insect species tested (Anticarsiagemmatalis, Pseudoplusia includes, and Spodoptera frugiperda) 3 leafpunches were made from each plant for a total of 45 leafdiscs/plant/insect species. The 1.4 cm diameter (or 1.54 cm²) leafpunches were placed in a test arena on top of 2% water agar, infestedwith one neonate larvae and sealed with a perforated plastic lid.Mortality and leaf consumption were rated 4 days after infestation.Larvae that were not responsive to gentle probing were considered dead.Leaf damage was assessed by visually scoring the percentage of leafpunch consumed by the insect.

Field evaluations were conducted by collecting leaf samples from seedincrease nursery plots in Santa Isabel, Puerto Rico and sending theseleaves to Indianapolis, Ind. for testing. The nursery plot for soybeanevent pDAB9582.814.19.1 was planted in February 2011 and consisted ofapproximately 180 plants arranged in four rows. Each row was 2.3 m longand spaced 76.2 cm apart; individual plants were spaced 5.1 cm apartwithin each row. In March 2011, one fully-expanded, mainstem trifoliateleaf, located approximately four nodes below the meristem, was excisedfrom 10 soybean event pDAB9582.814.19.1 plants and 10 ‘Maverick’ plants.The leaves were placed in labeled plastic bags, (one per bag) andsealed. The bagged leaves were packed and transferred to the laboratory.In the laboratory, one or two 3.33 cm (1.31 in) diameter leaf discs werepunched from each trifoliate leaf to provide a total of 16 leaf discs.Each leaf disc was placed a in test arena on top of 2% agar, infestedwith one neonate S. frugiperda larva, and sealed with a perforatedplastic lid. The leaf discs were held in a controlled environmentchamber for 7 days, at which time mortality and leaf consumption wererated. Larvae not responsive to gentle probing were considered dead.Leaf damage was assessed by visually scoring the percentage of leafpunch consumed by the insect.

The results obtained from these replicated experiments indicated thesoybean event pDAB9582.814.19.1 sustained significantly lower damagethan the Maverick control plants for all insects tested. Thus, thesoybean event pDAB9582.814.19.1 has insecticidal activity over thisbroad host range.

Example 7 Sequence of Soybean Event pDAB9582.814.19.1

SEQ ID NO:14 provides the sequence of soybean event pDAB9582.814.19.1.This sequence contains the 5′ genomic flanking sequence, the T-strandinsert of pDAB9582 and 3′ genomic flanking sequences. With respect toSEQ ID NO:14, residues 1-1400 are 5′ genomic flanking sequence, residues1401-1536 are residues of a rearrangement from the pDAB9582 plasmid and1537-13896 are residues of the pDAB9582 T-strand insert, and residues13897-15294 are 3′ flanking sequence. The junction sequence ortransition with respect to the 5′ end of the insert thus occurs atresidues 1400-1401 of SEQ ID NO:14. The junction sequence or transitionwith respect to the 3′ end of the insert thus occurs at residues13896-13897 of SEQ ID NO:14.

It should be noted that progeny from soybean event pDAB9582.814.19.1 mayhave sequences which slightly deviate from SEQ ID NO:14. During theintrogression and breeding process of introducing soybean eventpDAB9582.814.19.1 into the genome of plant cells, it is not uncommon forsome deletions or other alterations of the insert to occur. Moreover,errors in PCR amplification can occur which might result in minorsequencing errors. For example, flanking sequences listed herein weredetermined by generating amplicons from soybean genomic DNAs, and thencloning and sequencing the amplicons. It is not unusual to find slightdifferences and minor discrepancies in sequences generated anddetermined in this manner, given the many rounds of amplification thatare necessary to generate enough amplicon for sequencing from genomicDNAs. One skilled in the art should recognize and be put on notice thatany adjustments needed due to these types of common sequencing errors ordiscrepancies are within the scope of the subject invention. Thus, therelevant segment of the plasmid sequence provided herein might comprisesome minor variations. Thus, a plant comprising a polynucleotide havingsome range of identity with the subject insert sequence is within thescope of the subject invention. Identity to the sequence of SEQ ID NO:14can be a polynucleotide sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequenceexemplified or described herein. Thus, some differences between SEQ IDNO:14 and soybean event pDAB9582.814.19.1 progeny plants may beidentified and are within scope of the present invention.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

1. A polynucleotide comprising SEQ ID NO:14.
 2. A soybean plant, seed,or other part of said plant comprising event 814 as present in seeddeposited with the American Type Culture Collection under Accession No.PTA-12006.
 3. An isolated polynucleotide that is diagnostic for soybeanevent 9582.814.19.1, wherein said polynucleotide comprises SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:14, DNA molecules at least 25 base pairs inlength comprising base pairs 1400/1401 of SEQ ID NO:1 in each directionfrom the base pairs 1400/1401 junction; DNA molecules at least 10 basepairs in length comprising 1400/1401 of SEQ ID NO:1 in each directionfrom the base pairs 1400/1401 junction; amplicons at least 25 base pairsin length comprising 152-153 of SEQ ID NO:2 and at least 10 base pairsof SEQ ID NO:2 in each direction from the base pairs 152/153 junction.4. The isolated polynucleotide in claim 3 comprising one or moresequences selected from from the group consisting of base pairs1385-1415 of SEQ ID NO:1, base pairs 1350-1450 of SEQ ID NO:1, basepairs 1300-1500 of SEQ ID NO:1, base pairs 1200-1600 of SEQ ID NO:1,base pairs 137-168 of SEQ ID NO:2, base pairs 103-203 of SEQ ID NO:2,base pairs 3-303 of SEQ ID NO:2, and SEQ ID NO:14.
 5. A method ofdetecting the presence of soybean event 9582.814.19.1 in a sample, saidmethod comprising: a. providing a polynucleotide probe selected from thegroup consisting of base pairs 1385-1415 of SEQ ID NO:1, base pairs1350-1450 of SEQ ID NO:1, base pairs 1300-1500 of SEQ ID NO:1, basepairs 1200-1600 of SEQ ID NO:1, base pairs 137-168 of SEQ ID NO:2, basepairs 103-203 of SEQ ID NO:2, base pairs 3-303 of SEQ ID NO:2, and SEQID NO:14 or their complements thereof; b. isolating the genomic DNA ofthe sample; c. conducting stringent hybridization assay for thepolynucleotide probe in step a and the DNA in step b; and d. identifyingpositive hit as the presence of soybean event 9582.814.19.1 in thesample.
 6. A method of detecting the presence of soybean event9582.814.19.1 in a sample, comprising conducting a PCR amplification ofa. partial or all of the flanking sequence, and b. partial or all of theinsert sequence of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:14.
 7. Themethod of claim 6, wherein the first primer is a polynucleotide at least10 base pairs in length that selectively binds to a flanking sequencewithin residues 1-1400 of SEQ ID NO:1 or the complement thereof, and thesecond primer is selected from a polynucleotide at least 10 base pairsin length that selectively binds to an insert sequence within residues1401-1836 of SEQ ID NO:1.
 8. The method of claim 6, wherein the firstprimer is a polynucleotide at least 10 base pairs in length thatselectively binds to an insert sequence within residues 1-152 of SEQ IDNO:2 or the complement thereof, and the second primer is selected from apolynucleotide at least 10 base pairs in length that selectively bindsto a flanking sequence within residues 153-1550 of SEQ ID NO:2.